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Role of CHRAC14 and histone methyltransferase Su(var)3-9 in facilitating ectopic loading of CENP-A

Sharma, Abhishek

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Abstract

Kinetochore formation is required for the attachment of microtubules to the chromosome at centromere where CENP-A (also known as CID in Drosophila) is the key factor for the formation of kinetochore. CENP-A is a variant of histone H3 and its accumulation at the centromere leads to the initiation of kinetochore formation. CENP-A localization to centromere in Drosophila requires CENP-C and CAL1. Ectopic localization of CENP-A has been reported to give rise to pseudo-kinetochore, which results in chromosome segregation defect and could lead to aneuploidy. Breast, prostate and colon cancer show higher level of CENP-A with ectopic localization. In 2014 our lab reported that depletion of CHRAC14 leads to ectopic localization in Drosophila cells. CHRAC14 is a subunit of two chromatin remodelling complex, CHRAC and ATAC complex.

In this study, we have investigated the potential role of CHRAC14 in maintaining chromatin structure and how it’s absence lead to CENP-A ectopic localization. We show that depletion of CHRAC14 leads to increase in transcript level of histone methyltransferase Su(var)3-9. Su(var)3-9 is responsible for histone H3 lysine 9 (H3K9) methylation. Using Immunoblotting and immunofluorescence we show that lack of CHARC14 leads to overall increase in histone H3 lysine (H3K9) di methylation and reduction in H3K9 acetylation, thereby highlighting the importance of CHRAC14 in maintaining heterochromatin and euchromatin balance.

Further, we examined if the increase in Su(var)3-9 expression level could lead to ectopic CENP-A localization. Using Su(var)3-9 overexpressing Drosophila S2 cells, we show that overexpression of Su(var)3-9 can cause ectopic localization of CENP-A and CENP-C. Moreover, live-cell imaging analysis revealed that Su(var)3-9 overexpressing cells show cell segregation defect with delayed metaphase and lagging chromosome. Further analysis of the observed lagging chromosomes in Su(var)3-9 overexpressing cells revealed presence of higher number of CENP-A foci, indicating it to be the cause of cell segregation defect. Overall, this study reveals the role of CHRAC14 in maintaining the chromatin structure and highlights the significance of maintaining the physiological level of Su(var)3-9 for proper CENP-A localization.

Item Type: Dissertation
Supervisor: Schiebel, Prof. Dr. Elmar
Place of Publication: Heidelberg, Germany
Date of thesis defense: 26 January 2017
Date Deposited: 06 Feb 2017 11:23
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Controlled Keywords: Heterochromatin, CENP-A, Drosophila, Su(var)3-9
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